Pharmaceutical compositions comprising monoterpenes

ABSTRACT

The present invention provides a process for purifying a monoterpene or sesquiterpene having a purity greater than about 98.5% (w/w). The process comprises the steps of derivatizing the monoterpene (or sesquiterpene) to produce a monoterpene (or sesquiterpene) derivative, separating the monoterpene (or sesquiterpene) derivative, and releasing the monoterpene (or sesquiterpene) from the derivative. Also encompassed by the scope of the present invention is a pharmaceutical composition comprising a monoterpene (or sesquiterpene) having a purity greater than about 98.5% (w/w). The purified monoterpene can be used to treat a disease such as cancer. The present monoterpene (or sesquiterpene) may be administered alone, or may be co-administered with radiation or other therapeutic agents, such as chemotherapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/939,834 filed Jul. 11, 2013, now U.S. Pat. No. 9,133,085, issued Sep.15, 2015, which is a continuation of U.S. application Ser. No.13/040,059 filed Mar. 3, 2011, now U.S. Pat. No. 8,507,734, issued Aug.13, 2013 which claims priority to U.S. Provisional Application No.61/310,231 filed Mar. 3, 2010, the disclosures of which are incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to monoterpene or sesquiterpenecompositions. In particular, the present invention relates to usingmonoterpenes (such as (S)-perillyl alcohol) or sesquiterpenes having apurity greater than about 98.5% (w/w) to treat nervous system tumors.

BACKGROUND OF THE INVENTION

Malignant gliomas, the most common form of central nervous system (CNS)cancers, are currently considered essentially incurable. Among thevarious malignant gliomas, anaplastic astrocytomas (Grade III) andglioblastoma multiforme (GBM; Grade IV) have an especially poorprognosis due to their aggressive growth and resistance to currentlyavailable therapies. The present standard of care for malignant gliomasconsists of surgery, ionizing radiation, and chemotherapy. Despiterecent advances in medicine, the past 50 years have not seen anysignificant improvement in prognosis for malignant gliomas. Wen et al.Malignant gliomas in adults. New England J Med. 359: 492-507, 2008.Stupp et al. Radiotherapy plus concomitant and adjuvant temozolomide forglioblastoma. New England J Med. 352: 987-996, 2005.

A major reason for the poor prognosis of malignant gliomas is thedifficulty in delivering a sufficient quantity of chemotherapeuticagents to the brain. Drug access to the brain is limited by the bloodbrain barrier (BBB). The concentration of drugs that finally reach thebrain is further decreased by hepatic first-pass metabolism and urinaryexcretion. Therefore, invasive surgeries are often required, such astumor resection, stereotactic injection of anti-tumor medication, orplacement of catheters for convection enhanced delivery of medication.

Intranasal delivery of a drug offers a novel non-invasive therapy tobypass the blood brain barrier and to rapidly deliver pharmaceuticalagents to the CNS directly. Intranasally administered drugs reach theparenchymal tissues of the brain, spinal cord and/or cerebrospinal fluid(CSF) within minutes. In addition to delivery via the olfactory tractand trigeminal nerves, it appears from animal studies that thetherapeutic drug is also delivered systemically through the nasalvasculature. Hashizume et al. New therapeutic approach for brain tumors:intranasal delivery of telomerase inhibitor GRN163. Neuro-oncology 10:112-120, 2008. Thorne et al. Delivery of insulin-like growth factor-1 tothe rat brain and spinal cord along olfactory and trigeminal pathwaysfollowing intranasal administration. Neuroscience 127: 481-496, 2004.Intranasal delivery of therapeutic agents may provide a systemic methodfor treating other types of cancers, such as lung cancer, prostatecancer, breast cancer, hematopoietic cancer and ovarian cancer, etc.

Perillyl alcohol (POH), a naturally occurring monoterpene, has beensuggested to be an effective agent against a variety of cancers,including CNS cancer, breast cancer, pancreatic cancer, lung cancer,melanomas and colon cancer. Gould, M. Cancer chemoprevention and therapyby monoterpenes. Environ Health Perspect. 1997 June; 105 (Suppl 4):977-979. Oral perillyl alcohol has been used in a recent phase I trialsponsored by the National Cancer Institute. Although oral perillylalcohol did not induce severe adverse effects, it was generally poorlytolerated, mainly due to gastrointestinal side effects. In addition, itsanti-cancer efficacy was limited. As a result, the use of oral perillylalcohol was discontinued. Ripple et al. Phase I clinical andpharmacokinetic study of perillyl alcohol administered four times a day.Clinical Cancer Res 6: 390-6, 2000.

In order to minimize the gastrointestinal side effects of oral POH andto provide a means of delivering POH directly to the central nervoussystem, a nasal formulation of POH (see below) for direct intranasaldelivery of POH to malignant brain tumors was studied by Dr. ClovisFonseca at the Fluminese University in Brazil. Da Fonseca, et al.Anaplastic oligodendroglioma responding favorably to intranasal deliveryof perillyl alcohol: a case report and literature review, SurgicalNeurology (2006) 66:611-615. This formulation of commercial grade POHcombined with a solvent cocktail, has already been delivered to 150patients with recurrent malignant gliomas, with minimal side effect anda six month 50% progression free survival rate. Da Fonseca et al.Correlation of tumor topography and peritumoral edema of recurrentmalignant gliomas with therapeutic response to intranasal administrationof perillyl alcohol. Invest New Drugs 2009, Jan. 13.

Commercial grade perillyl alcohol, with purities ranging from 85% to96%, is typically purified from natural products, or by syntheticallymodifying natural products such as beta-pinene (extracted from pinetrees). Inevitably, perillyl alcohol obtained through these routes iscontaminated by its isomers and other impurities which have similarphysicochemical properties, and, therefore, are extremely difficult toremove from perillyl alcohol by conventional purification methods suchas fractional distillation or chromatography. Isomers of perillylalcohol and other impurities may be potentially inhibitory towards thedesired therapeutic properties of perillyl alcohol.

Consequently, there is still a need to prepare highly purified perillylalcohol and use this material in the treatment of CNS cancers such asmalignant gliomas, as well as other aggressive brain tumors. Purifiedperilly alcohol may be administered alone or in combination with othertreatment methods including radiation, standard chemotherapy, andsurgery. The administration can also be through various routes includingintranasal, oral, oral-tracheal for pulmonary delivery, and transdermal.

SUMMARY OF THE INVENTION

The present invention provides for a process of purifying (S)-perillylalcohol comprising the steps of: (a) derivatizing a mixture comprising(S)-perillyl alcohol to form a perillyl alcohol derivative, wherein theperillyl alcohol derivative has at least one property that allows it tobe separated from the mixture; (b) separating the perillyl alcoholderivative from the mixture using the property for separation; (c)releasing the (S)-perillyl alcohol from the perillyl alcohol derivativefrom step (b); and, (d) isolating the (S)-perillyl alcohol from step(c). The (S)-perillyl alcohol has a purity greater than about 98.5%(w/w), greater than about 99.0% (w/w), or greater than about 99.5%(w/w). In certain embodiments, the mixture further comprisesnatural-product-derived or other impurities. The property of theperillyl alcohol derivative can be to form crystals, and the separationin step (b) can, therefore, be through crystallization. The separationin step (b) may also be through chromatography. The perillyl alcoholderivative can be a perillyl alcohol ester. In one embodiment, theperillyl alcohol ester is a benzoate ester, such as 3,5-dinitrobenzoateester.

The invention also encompasses an (S)-perillyl alcohol, where the(S)-perillyl alcohol has a purity greater than about 98.5% (w/w),greater than about 99.0% (w/w), or greater than about 99.5% (w/w).

The invention further provides for a pharmaceutical compositioncomprising (S)-perillyl alcohol having a purity greater than about 98.5%(w/w). The (S)-perillyl alcohol may have a purity greater than about98.5% (w/w). The pharmaceutical composition may contain from about 0.1%(w/w) to about 100% (w/w) (S)-perillyl alcohol. In addition, thepharmaceutical composition may comprise a chemotherapeutic agent, aswell as at least one pharmaceutically acceptable excipient. Thechemotherapeutic agent may be a DNA alkylating agent, a topoisomeraseinhibitor, an endoplasmic reticulum stress inducing agent, a platinumcompound, an antimetabolite, an enzyme inhibitor, a receptor antagonist,a therapeutic antibody, or a vaccine. In certain embodiments, thechemotherapeutic agent is dimethyl-celecoxib (DMC), irinotecan (CPT-11),temozolomide, or rolipram. The pharmaceutical composition can beadministered alone, or may be administered before, during or afterradiation, or before, during or after the administration of achemotherapeutic agent. The routes of administration include inhalation,intranasal, oral, intravenous, subcutaneous and intramuscular injection.The pharmaceutical composition can be administered intranasally by anintranasal spray device, an atomizer, a nebulizer, a metered doseinhaler (MDI), a pressurized dose inhaler, an insufflator, an intranasalinhaler, a nasal spray bottle, a unit dose container, a pump, a dropper,a squeeze bottle, or a bi-directional device. The pharmaceuticalcomposition may be administered intranasally in the form of a gel, anointment, a nasal emulsion, a lotion, a cream, a nasal tampon, or abioadhesive strip.

The present invention further provides for a method of treating cancer,comprising the step of delivering to a mammal a therapeuticallyeffective amount of (S)-perillyl alcohol having a purity greater thanabout 98.5% (w/w). The (S)-perillyl alcohol may be admixed orcoformulated with a therapeutic agent, for example, a chemotherapeuticagent. The cancer may be a tumor of the nervous system, such as aglioblastoma, or other tumors.

The present invention provides for an article of manufacture (e.g., akit) comprising (S)-perillyl alcohol formulated for intranasaladministration, and a device for intranasal administration of the(S)-perillyl alcohol, wherein the (S)-perillyl alcohol has a purity ofgreater than about 98.5% (w/w). The device may be an intranasal spraydevice, an atomizer, a nebulizer, a metered dose inhaler (MDI), apressurized dose inhaler, an insufflator, an intranasal inhaler, a nasalspray bottle, a unit dose container, a pump, a dropper, a squeezebottle, or a bi-directional device. The article of manufacture mayfurther comprise printed matter which states the (S)-perillyl alcohol isto be used to treat cancer, such as glioblastoma. The printed matter mayfurther state the (S)-perillyl alcohol is to be administered alone, oradministered in combination with radiation, surgery or chemotherapeuticagents.

Also provided for is a method of inhibiting the growth of a cell,comprising the step of contacting the cell with an effective amount of(S)-perillyl alcohol having a purity greater than about 98.5% (w/w). Thecontacting may occur in vitro or in vivo. The cell may be a glioma cell,a meningioma cell, a pituitary adenoma cell, a lung cancer cell, aprostate cancer cell, a breast cancer cell, a hematopoietic cancer cell,a melanoma cell, or an ovarian cancer cell. The cell may be atemozolomide-resistant cell or a cancer stem cell.

The compositions and methods of the present invention may be used todecrease or inhibit angiogenesis. The present compositions and methodsmay decrease or inhibit production of pro-angiogenic cytokines,including, but not limited to, vascular endothelial growth factor (VEGF)and interleukin 8 (IL8).

The compositions and methods of the present invention may be used toincrease paracellular permeability, for example, paracellularpermeability of endothelial cells or epithelial cells. The presentcompositions and methods may be used to increase blood brain barrierpermeability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows light microscopic images demonstrating morphologicalchanges of human malignant glioma cells A172 after being treated withpurified (S)-perillyl alcohol having a purity greater than 98.5%(hereinafter “purified POH” or “purified (S)-perillyl alcohol”), as wellas (S)-perillyl alcohol purchased from Sigma Chemicals having a purityof about 96%. Increased cytotoxicity is observed in the A172 gliomacells after treatment with purified (S)-perillyl alcohol (FIG. 1C)compared to commercial (S)-perillyl alcohol from Sigma Chemicals (FIGS.1A and 1B).

FIG. 2 shows the results of the MTT cytotoxicity assays performed onhuman malignant glioma cells U87 with purified (S)-perillyl alcoholhaving a purity greater than 98.5% compared to commercial grade(S)-perillyl alcohol from Sigma Chemicals having a purity of about 96%.Cells treated with phosphate-buffered saline (PBS) were used as control.The difference in cytotoxic effect between the purified (S)-perillylalcohol and Sigma (S)-perillyl alcohol is much greater than thedifference in (S)-perillyl alcohol purity. See details in the Examplesbelow.

FIG. 3A shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of purified POH in killing U251, U87, LN299 and A172 humanglioma cells.

FIG. 3B shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of purified POH in killing U251 human glioma cells(temozolomide-sensitive) and U251 temozolomide-resistant cells. U251/TR1and U251/TR2 refer to two temozolomide-resistant U251 cell lines.

FIG. 3C shows the results of the colony formation assays performed onU251 human glioma cells (temozolomide-sensitive) and U251temozolomide-resistant cells (U251/TR1).

FIGS. 4A and 4B show the results of the colony formation assaysperformed on A172 (FIG. 4A), U251 human malignant glioma cells, andB16-F1 melanoma cells (FIG. 4B) treated with purified (S)-perillylalcohol having a purity greater than 98.5% alone, radiation only, orpurified (S)-perillyl alcohol plus radiation. In all three cell lines,synergistic cytotoxicity was achieved by the combination of purified(S)-perillyl alcohol plus radiation.

FIG. 5A shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of DMC or DMC combined with purified POH in killing U251,LN229 and U87 human glioma cells.

FIG. 5B shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of Nelfinavir (NFV) or NFV combined with purified POH inkilling U251 human glioma cells.

FIG. 5C shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of TMZ or TMZ combined with purified POH in killing U251human glioma cells.

FIG. 6 shows the results of propidium iodide (PI) staining of A172,A375, U251 human malignant glioma cells and B16-F1 melanoma cells afterbeing treated with purified (S)-perillyl alcohol having a purity greaterthan 98.5%, demonstrating that (S)-perillyl alcohol increasesparacellular permeability.

FIG. 7A shows the experimental scheme of the transepithelial electricalresistance (TEER) measurement to assess paracellular permeability.

FIG. 7B shows that (S)-perillyl alcohol decreases the TEER,corresponding to an increase in paracellular permeability. In theseexperiments, brain endothelial cells (BECs) were used as an in vitromodel of blood brain barrier. The increase in TEER in each group overtime corresponds to increased confluency of the cells in culture. TEERdecreased when cells were treated by 0.03% POH for 20 hours compared tomedium only, 0.01% POH treatment, and 0.02% POH treatment.

FIG. 8 shows that (S)-perillyl alcohol increases epithelial paracellularpermeability. Madin-Darby Canine Kidney (MDCK) epithelial cells in amonolayer were treated overnight with varying concentrations of purified(S)-perillyl alcohol having a purity greater than 98.5%. Fluoresceinlabeled antibodies were then added to the cells. The amount of labeledantibodies that crossed the cell monolayer was then quantitated byfluorescence.

FIG. 9 shows the results of ELISA assays demonstrating that POHtreatment decreases cytokine IL-8 (FIG. 9A) and VEGF secretion (FIG. 9B)in U87 human glioma cells. ELISA was performed after cells were treatedwith POH for 48 hours.

FIG. 10 shows Western blot results demonstrating that POH inducesendoplasmic reticulum (ER) stress. POH up-regulated the levels of ERstress marker glucose-regulated protein 78 (GRP78) and apoptosis markereCCAAT/enhancer binding protein (CHOP) in U251, U87 and A172 human gliomacells. The treatments for different lanes of the Western blot are asfollows: 1: No treatment; 2: 0.5 mM purified POH; 3: 1.5 mM purifiedPOH; 4: 0.5 mM Sigma POH; 5: 1.5 mM Sigma POH; 6: 0.5 mM Wako POH; 7:1.5 mM Wako POH; 8: Positive Control (DMC 40 uM). Sigma POH is the POHpurchased from Sigma Chemicals. Wako POH is the POH purchased from SigmaChemicals. Cells were treated with different agents or mock treated for20 hours before being lysed.

FIG. 11 shows Western blot performed after U251 TMZ-sensitive andTMZ-resistant (U251/TR1, U251/TR2) cells were treated for 20 hours withSigma POH (1.5 mM; “S”), purified (S)-perillyl alcohol (1.5 mM; “G”), ornot treated (control; “C”). The results demonstrate increased levels ofglucose-regulatory protein 78 (GRP-78) and the apoptosis marker CHOP,showing increased endoplasmic reticulum (ER) stress after treatment.Sigma POH is the POH purchased from Sigma Chemicals.

FIG. 12 shows Western blot performed after U251 glioma cells weretreated for 24 hours with Sigma POH or purified POH. The resultsdemonstrate that POH treatment decreases K-Ras and H-Ras expression.

FIG. 13 shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of POH in killing USC-02 glioblastoma cancer stem cellline. The cancer stem cells were more sensitive to POH than glioma tumorcells (U251 cells) or normal stem cells (mSVZ: mouse SVZ stem/progenitorcells).

FIG. 14 shows the results of the MTT cytotoxicity assays demonstratingthat the glioma tumor cells (U251 cells) were more sensitive to POH thannormal brain cells (brain endothelial cells (BECs) and astrocytes).

FIG. 15A shows the results of the MTT cytotoxicity assays demonstratingthat the cancer stem cells USC-04 were more sensitive to POH than gliomatumor cells (U251 cells).

FIG. 15B shows the results of the MTT cytotoxicity assays demonstratingthat the cancer stem cells USC-04 were more resistant to DMC than gliomatumor cells (U251 cells). Verapamil is used as an inhibitor of drugefflux pump proteins such as P-glycoprotein. The results suggest thatDMC resistance of the cancer stem cells was not due to drug efflux viaP-glycoprotein.

FIG. 16 shows the results of sphere formation assay (SFA) demonstratingthat POH decreased the number of spheres formed in glioblastoma cancerstem cells (USC04). USC04 cells were incubated with varyingconcentrations of POH (0-1.5 mM) for 6 days.

FIG. 17 shows that POH decreased H-ras production in glioblastoma cancerstem cells. Two glioblastoma cancer stem cell lines (USC-02, USC-04)were treated by POH for 24 hours. USC-02 and USC-04 are two independentprimary cancer stem cell lines isolated from glioblastoma tissue fromtwo different patients.

FIG. 18 shows comparison of POH IC50 values on various cell lines.Cancer stem cells (USC-02 cells) are the most sensitive to POH among thecell lines tested. Normal cells, including normal stem cells mSVZ (mouseSVZ stem cells), brain endothelial cells (BECs) and astrocytes, are themost resistant to POH.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for methods of purifying perillyl alcoholfrom its isomers (including enantiomer) and other impurities thattypically accompany perillyl alcohol when it is produced from naturalproducts and/or synthetic sources. Perillyl alcohol may be purified byderivatizing perillyl alcohol to produce a crystalline derivative suchas its 3,5-dinitrobenzoate ester. The perillyl alcohol derivative canthen be separated from its accompanying contaminants (whether or not thecontaminants are also present as derivatives or not) by suitabletechniques, such as conventional crystallization, or preparativechromatography. The purified perillyl alcohol derivative can then beconverted to perillyl alcohol which has a purity greater than about98.5% (w/w). The purified perillyl alcohol may be administered to asubject alone, or may be co-administered together with other agents. Forexample, the purified perillyl alcohol may be used to sensitize a cancerpatient to radiation or chemotherapy. Compared to commercially available(S)-perillyl alcohol, the purified (S)-perillyl alcohol demonstratesdisproportionately enhanced activity in cellular assays and othertherapeutic test models.

The present invention provides for a process of preparing a purifiedform of a monoterpene or sesquiterpene. The monoterpene (orsesquiterpene) is purified by the following steps: (a) derivatizing amixture comprising monoterpene (or sesquiterpene) to form a monoterpene(or sesquiterpene) derivative, wherein the monoterpene (orsesquiterpene) derivative has at least one property that allows it to beseparated from the mixture; (b) separating the monoterpene (orsesquiterpene) derivative from the mixture using the property forseparation; (c) releasing the monoterpene (or sesquiterpene) from themonoterpene (or sesquiterpene) derivative from step (b); and, (d)isolating the monoterpene (or sesquiterpene) from step (c). The purifiedmonoterpene (or sesquiterpene) may have a purity greater than about98.5% (w/w), about 99.0% (w/w), or about 99.5% (w/w). In certainembodiments, the mixture further comprises natural-product-derived orother impurities.

The property of the monoterpene (or sesquiterpene) derivative can be toform crystals, and the separation in step (b) can, therefore, be throughcrystallization. The monoterpene (or sesquiterpene) is purified by thefollowing steps: (a) derivatizing the monoterpene (or sesquiterpene) toform a monoterpene (or sesquiterpene) derivative; (b) crystallizing themonoterpene (or sesquiterpene) derivative; (c) separating themonoterpene (or sesquiterpene) derivative crystals of step (b); (d)converting the separated monoterpene (or sesquiterpene) derivative tomonoterpene (or sesquiterpene); and (e) isolating the monoterpene (orsesquiterpene).

The separation of the monoterpene (or sesquiterpene) derivative from themixture may also be through other suitable separation techniques knownin the art, including, but not limited to, chromatography, adsorption,centrifugation, decantation, distillation, electrophoresis, evaporation,extraction, flotation, filtration, precipitation, sedimentation.Wikipedia—Separation Process. Retrieved on Feb. 11, 2010 from URL:http://en.wikipedia.org/wiki/Separation_of_mixtures. The property of themonoterpene (or sesquiterpene) derivative useful for separation of thederivative from the mixture can be any of its physicochemical propertiesthat are different from that of the other components in the mixture. Thephysicochemical properties include, but are not limited to, solubility,polarity, partition coefficient, affinity, size, hydrodynamic diameter,and charge. The monoterpene (or sesquiterpene) derivative can beprepared where the derivative has at least one different property thanthat of its isomers, structural variants, or contaminants present in thestarting material. The chromatography can be any suitable preparativechromatography, including, but not limited to, gas chromatography (GC),high pressure liquid chromatography (HPLC), affinity chromatography, ionexchange chromatography, size exclusion chromatography, andreversed-phase chromatography.

In one embodiment, the monoterpene may be (S)-perillyl alcohol, and thederivatization reaction can involve esterification. For example,(S)-perillyl alcohol may be prepared using a 3,5-dinitrobenzoate esterderivative.

The present invention further provides for a monoterpene (orsesquiterpene) composition having a purity of greater than about 98.5%(w/w), greater than about 99.0% (w/w), or greater than about 99.5%(w/w).

The purified monoterpene (or sesquiterpene) may be formulated into apharmaceutical composition, where the monoterpene (or sesquiterpene) ispresent in amounts ranging from about 0.01% (w/w) to about 100% (w/w),from about 0.1% (w/w) to about 80% (w/w), from about 1% (w/w) to about70% (w/w), from about 10% (w/w) to about 60% (w/w), or from about 0.1%(w/w) to about 20% (w/w). In addition, the pharmaceutical compositionmay contain a therapeutic agent, such as a chemotherapeutic agent. Thetherapeutic agent may be dissolved in perillyl alcohol. The presentcompositions can be administered alone, or may be co-administeredtogether with radiation or another agent (e.g., a chemotherapeuticagent), to treat a disease such as cancer. Treatments may be sequential,with the monoterpene (or sesquiterpene) being administered before orafter the administration of other agents. Alternatively, agents may beadministered concurrently. The route of administration may vary, and caninclude, inhalation, intranasal, oral, transdermal, intravenous,subcutaneous or intramuscular injection.

The present invention also provides for a method of treating a diseasesuch as cancer, comprising the step of delivering to a patient atherapeutically effective amount of a purified monoterpene (orsesquiterpene) prepared by the methods of the present invention.

The compositions of the present invention may contain one or more typesof monoterpene (or sesquiterpene). Monoterpenes include terpenes thatconsist of two isoprene units and have the molecular formula C₁₀H₁₆.Monoterpenes may be linear (acyclic) or contain rings. Monoterpenoids,produced by biochemical modifications such as oxidation or rearrangementof monoterpenes, and pharmaceutically acceptable salts of monoterpenesor monoterpenoids, are also encompassed by the present invention.Examples of monoterpenes and monoterpenoids include, perillyl alcohol(S(−)) and R(+)), geranyl pyrophosphate, ocimene, myrcene, geraniol,citral, citronellol, citronellal, linalool, pinene, terpineol, terpinen,limonene, terpinenes, phellandrenes, terpinolene, terpinen-4-ol (or teatree oil), pinene, terpineol, terpinen; the terpenoids such as p-cymenewhich is derived from monocyclic terpenes such as menthol, thymol andcarvocrol; bicyclic monoterpenoids such as camphor, borneol andeucalyptol.

Monoterpenes may be distinguished by the structure of a carbon skeletonand may be grouped into acyclic monoterpenes (e.g., myrcene, (Z)- and(E)-ocimene, linalool, geraniol, nerol, citronellol, myrcenol, geranial,citral a, neral, citral b, citronellal, etc.), monocyclic monoterpenes(e.g., limonene, terpinene, phellandrene, terpinolene, menthol, carveol,etc.), bicyclic monoterpenes (e.g., pinene, myrtenol, myrtenal,verbanol, verbanon, pinocarveol, carene, sabinene, camphene, thujene,etc.) and tricyclic monoterpenes (e.g. tricyclene). See Encyclopedia ofChemical Technology, Fourth Edition, Volume 23, page 834-835.

Sesquiterpenes of the present invention include terpenes that consist ofthree isoprene units and have the molecular formula C₁₅H₂₄.Sesquiterpenes may be linear (acyclic) or contain rings.Sesquiterpenoids, produced by biochemical modifications such asoxidation or rearrangement of sesquiterpenes, are also encompassed bythe present invention. Examples of sesquiterpenes include farnesol,farnesal, farnesylic acid and nerolidol.

The purified monoterpene (or sesquiterpene) is prepared using thederivatized monoterpene (or sesquiterpene), which may be separated fromits accompanying contaminants (such as its isomers) by crystallization.The crystallization and purification may also enhance the chiral purityof the monoterpene (or sesquiterpene).

The derivatives of monoterpene (or sesquiterpene) include, but are notlimited to, esters, alcohols, aldehydes and ketones of the monoterpene(or sesquiterpene). Monoterpene (or sesquiterpene) alcohols may bederivatized to esters, aldehydes or acids. The derivatives ofmonoterpene (or sesquiterpene) can be used to regenerate the monoterpene(or sesquiterpene) through chemical reactions known to a person skilledin the art. For example, an ester of a monoterpene (or sesquiterpene)can be hydrolyzed to generate the monoterpene (or sesquiterpene).

In one embodiment, a monoterpene (or sesquiterpene) is purified using anester of the monoterpene (or sesquiterpene). The purification processincludes the following steps: (a) derivatizing a monoterpene (orsesquiterpene) to produce an ester of the monoterpene (orsesquiterpene); (b) crystallizing the ester of the monoterpene (orsesquiterpene); (c) separating crystals of the ester of the monoterpene(or sesquiterpene) of step (b); (d) converting the ester of themonoterpene (or sesquiterpene) to the monoterpene (or sesquiterpene);and (e) isolating the monoterpene (or sesquiterpene).

Esters of the monoterpene (or sesquiterpene) alcohols of the presentinvention can be derived from an inorganic acid or an organic acid.Inorganic acids include, but are not limited to, phosphoric acid,sulfuric acid, and nitric acid. Organic acids include, but are notlimited to, carboxylic acid such as benzoic acid, fatty acid, aceticacid and propionic acid. Examples of esters of monoterpene (orsesquiterpene) alcohols include, but are not limited to, carboxylic acidesters (such as benzoate esters, fatty acid esters (e.g., palmitateester and linoleate ester), acetates, propionates (or propanoates), andformates), phosphates, sulfates, and carbamates (e.g.,N,N-dimethylaminocarbonyl). Wikipedia—Ester. Retrieved from URL:http://en.wikipedia.org/wiki/Ester.

In one embodiment, the derivatives are benzoate esters including, butnot limited to, 3,5-dinitrobenzoate ester, 4-nitrobenzoate ester,3-nitrobenzoate ester, 4-chlorobenzoate ester, 3,4,5-trimethoxybenzoateester and 4-methoxybenzoate ester, esters of hydroxybenzoic acid such asthe methyl, ethyl, propyl, isopropyl, butyl, isobutyl, hexyl, heptyl andbenzyl esters. (See, for example, Wikipedia—Benzoate esterhttp://commons.wikimedia.org/wiki/Category:Benzoate_esters).

A specific example of a monoterpene that may be used in the presentinvention is perillyl alcohol (commonly abbreviated as POH). Perillylalcohol compositions of the present invention can contain (S)-perillylalcohol, (R)-perillyl alcohol, or a mixture of (S)-perillyl alcohol and(R)-perillyl alcohol.

Perillyl alcohol may be purified by the following steps: (a)derivatizing a mixture comprising perillyl alcohol to form a perillylalcohol derivative, wherein the perillyl alcohol derivative has at leastone property that allows it to be separated from the mixture; (b)separating the perillyl alcohol derivative from the mixture using theproperty for separation; (c) releasing the perillyl alcohol from theperillyl alcohol derivative from step (b); and (d) isolating theperillyl alcohol from step (c). In certain embodiments, the mixturefurther comprises natural-product-derived or other impurities.

Perillyl alcohol may be purified using the methods of the presentinvention with perillyl alcohol derivatives. The derivatives include,perillyl alcohol esters, perillic aldehyde, dihydroperillic acid, andperillic acid. The derivatives of perillyl alcohol may also include itsoxidative and nucleophilic/electrophilic addition derivatives. U.S.Patent Publication No. 20090031455. U.S. Pat. Nos. 6,133,324 and3,957,856. Many examples of derivatives of perillyl alcohol are reportedin the chemistry literature (CAS Scifinder search output file, retrievedJan. 25, 2010).

In a specific example, perillyl alcohol is purified by: (a) derivatizingperillyl alcohol to produce a perillyl alcohol ester; (b) crystallizingthe perillyl alcohol ester; (c) separating the perillyl alcohol estercrystals of step (b) (e.g., from a mother liquor); (d) converting theseparated perillyl alcohol ester to generate perillyl alcohol; and (e)isolating the perillyl alcohol. The derivative of perillyl alcohol canbe used to regenerate the perillyl alcohol through chemical reactionsknown to a person skilled in the art. For example, an ester of perillylalcohol, such as a 3,5-dinitrobenzoate ester, can be hydrolyzed togenerate perillyl alcohol.

In certain embodiments, esters or ethers of perillyl alcohol may beprepared by reacting perillyl alcohol with acid chlorides or alkylchlorides, the chemical structures of which are shown below.

For the esterification reaction, the molar ratio of perillyl alcohol toacid chloride (or alkyl chloride) may range from about 1:1 to about 1:2,from about 1:1 to about 1:1.5, including, for example, about 1:1.05,about 1:1.1, about 1:1.2, about 1:1.3, or about 1:1.4.

Suitable reaction solvents include, but are not limited to,dichloromethane, diethyl ether, diisopropyl ether, and methyl-t-butylether. The reaction may be performed at a temperature ranging from about−5° C. to about 50° C., or from about −5° C. to 25° C. Suitable basesthat may be included in the reaction include, but are not limited to,organic bases, such as triethylamine, di-isopropylamine,N,N′-diisopropylethylamine, butylamine, sodium methoxide, potassiummethoxide, and potassium-t-butoxide. The esters thus generated are3,5-dinitrobenzoate ester, 4-nitrobenzoate ester, 3-nitrobenzoate ester,4-chlorobenzoate ester, 3,4,5-trimethoxybenzoate ester,4-methoxybenzoate ester and triphenylmethyl ester. The details of thechemical reactions are described in the Examples below.

The crystallizable monoterpene (or sesquiterpene) derivative may bepurified by crystallization or preparative chromatography.Crystallization separates a product from a liquid feedstream, often inextremely pure form, by cooling the feedstream or adding precipitantswhich lower the solubility of the desired product so that it formscrystals. For crystallization to occur, the solution must besupersaturated. This means that the solution has to contain moredissolved solute entities than it would contain under the equilibrium(saturated solution). This can be achieved by various methods, suchas 1) solution cooling; 2) addition of a second solvent to reduce thesolubility of the solute (a technique known as antisolvent ordrown-out); 3) chemical reaction; and 4) change in pH. Solventevaporation, spherical crystallization, fractional crystallization,fractional freezing procedures, and other suitable methods can also beused. Mersmann, A. Crystallization Technology Handbook. Edition 2(2001), published by CRC Press. Myerson et al. Crystallization As aSeparations Process (ACS Symposium Series) (1990), published by AmericanChemical Society.

In a specific example, crystallization of 3,5-dinitrobenzoate ester ofperillyl alcohol is carried out as follows. The aqueous layer containing3,5-dinitrobenzoate ester is extracted with dichloromethane and washedwith water. The organic layer which contains the 3,5-dinitrobenzoateester is dried over sodium sulphate. The organic layer is then filteredand concentrated. The resulting residue is finally crystallized from adiisopropyl ether mother liquor. A mother liquor is the part of a liquidthat is above the crystal solids, and, thus, can be separated from thecrystals. The separation of the crystals from the mother liquor can becarried out using any suitable techniques, including, but not limitedto, filtration (with or without the assistance of pressure and/orvacuum), centrifugation, and decantation.

Suitable solvents for crystallization of benzoate ester of perillylalcohol include, but are not limited to, ketone solvents (such asacetone, methyl ethyl ketone, methyl isobutyl ketone, n-butanone, andt-butylketone); nitrile solvents (such as acetonitrile, andpropionitrile); halogenated solvents (such as dichloromethane1,2-dichloroethane, and chloroform); esters (such as ethyl acetate,n-propylacetate, isopropyl acetate, and t-butylacetate); ethers (such asdiethyl ether, diisopropyl ether, methyl-t-butyl ether, tetrahydrofuranand 1,4-dioxane); hydrocarbon solvents (such as hexanes, cyclohexane,toluene and xylene); and mixtures thereof. In one embodiment, thesolvent contains methyl-t-butyl ether. The dissolution of benzoate esterin methyl-t-butyl ether (7-10 volumes) may be performed at an elevatedtemperature, if required, to achieve the desired concentration. Further,an activated charcoal treatment may be performed to remove coloredimpurities or to reduce the content of heavy metals, if any, or toremove any extraneous matter from the solution containing benzoateester. The crystallization from the resultant reaction mixture may becarried out by cooling the reaction mixture to a lower temperature ofabout 25° C. to about 0° C. Separation of the crystals may be carriedout by removal of the solvent followed by cooling the reaction mixture.Solvent may be removed by suitable techniques including evaporationusing a rotary evaporator, such as a Buchi rotavapor under vacuum.Crystals may be isolated from the reaction mixture by any conventionaltechnique such as filtration by gravity or by suction. In oneembodiment, the benzoate ester may be isolated by filtration and, ifdesired, may be further washed with a solvent. The benzoate ester may bedried by any of the conventional techniques such as drying in a traydryer, vacuum dryer, or air oven. The drying may be carried out at atemperature of about 30° C. to about 60° C. in a vacuum oven.

The purity of the crystallizable monoterpene (or sesquiterpene)derivative, and therefore, the purity of the monoterpene (orsesquiterpene), may be further improved by recrystallization. Varioustechniques can be used, such as single-solvent recrystallization,multi-solvent recrystallization, hot filtration-recrystallization, aswell as other suitable recrystallization techniques which are well knownin the art. Wikipedia—Recrystallization. Retrieved from URL:http://en.wikipedia.org/wiki/Recrystallization_(chemistry).

For example, U.S. Pat. No. Re. 32,241 describes an apparatus having acomponent crystallize on a cooled surface as material containing thecomponent flows theredown. U.S. Pat. No. 4,666,456 describes continuouspartial crystallization of a compound from a liquid mixture in which themixture is fed through a cascade of cooling sections. U.S. Pat. No.5,127,921 provides a multi-stage recrystallization procedure includingcontrolling reflux ratio conditions by regulating quantities of crystalsand mother liquor reflux materials.

The purity of the perillyl alcohol may be greater than about 98.5%(w/w), greater than about 99% (w/w), greater than about 99.5% (w/w), orgreater than about 99.9% (w/w).

In certain embodiments, the compounds of the invention contain one ormore chiral centers. The term “purity” can also encompass chiral purity.The purity of a stereoisomer of a monoterpene (or sesquiterpene) refersto chemical purity and/or chiral purity of the stereoisomer. Forexample, the purity of (S)-perillyl alcohol can include both thechemical purity and the chiral purity of (S)-perillyl alcohol. Thechiral purity of a stereoisomer of the monoterpene (or sesquiterpene)may be greater than about 98.5% (w/w), greater than about 99% (w/w),greater than about 99.5% (w/w), or greater than about 99.9% (w/w).

The chiral purity of (S)-perillyl alcohol may be greater than about98.5% (w/w), greater than about 99% (w/w), greater than about 99.5%(w/w), or greater than about 99.9% (w/w). In certain embodiments, thespecific optical rotation of (S)-perillyl alcohol of the presentinvention may range from −87.95° to −91.9°, when the specific opticalrotation is measured at 22° C. with the sample concentration at 1 g/mlin MeOH (see Table 1 for examples of specific optical rotation of(S)-perillyl alcohol).

TABLE 1 Estimated chiral purity of perillyl alcohol samples based onoptical rotation Purity by Specific Purity Neonc optical by analysisrotation Vendor (%) GC (C = 1, Sample Vendor Lot# Quantity (%) (by area)MeOH) description Wako ASK0744 5.0 g 85 90.4 −80.9° Wako feed stock WakoKWHO744 400 g 85 89.5 −81.5° Wako feed stock ( Neonc Sample# 12) AldrichMKAA4409 2 × 50 g 96 95.0 −88.7° Aldrich lab sample Aldrich MKAA0552 100g •90   96.2 −87.6° Aldrich bulk representative sample Neonc SGP-527-1.0 g 97.1 −88.2° Prepared from 130 KWHO744 (single ( Neonc crystallizedfrom Sample# 07) diisopropyl ether) Neonc SGP-527- 1.0 g 98.7 −87.9°Prepared from 133 KWHO744 ( Neonc (Double Sample# 09) recrystallizedfrom diisopropyl ether then from 2-propanol) Neonc SGP-527- 1.0 g 98.7−89.8° Prepared from 138 Aldrich ( Neonc MKAA0552 Sample# 10) NeoncSGP-527- 44.0 g 98.6 -91.9° Prepared from 153 Wako ( Neonc KWH0744Sample# 13) Neonc SGP-527- 46.0 g 98.6 -91.7° Prepared from 155 Wako (Neonc KWH0744 Sample# 14)

The purity of the monoterpene (or sesquiterpene) may be assayed by gaschromatography (GC) or high pressure liquid chromatography (HPLC). Othertechniques for assaying the purity of monoterpene (or sesquiterpene) andfor determining the presence of impurities include, but are not limitedto, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry(MS), GC-MS, infrared spectroscopy (IR), and thin layer chromatography(TLC). WHO Specifications and Evaluations for Public Health Pesticides:Malathion, World Health Organization, 2003. Chiral purity can beassessed by chiral GC or measurement of optical rotation.

Alternatively, the monoterpene (or sesquiterpene) may be purified bymethods other than crystallizing the derivates. For example, amonoterpene (or sesquiterpene) derivative can be prepared where thederivative has different physicochemical properties (e.g., solubility orpolarity) than that of its isomers, structural variants, or contaminantspresent in the starting material. Accordingly, the monoterpene (orsesquiterpene) derivative can be separated from the monoterpene (orsesquiterpene) by suitable separation techniques known in the art, suchas preparative chromatography.

The purified monoterpene (or sesquiterpene) may be stable after storage.For example, after storage at about 5•C for at least 3 months, thepresent composition may contain greater than about 98.5% (w/w), greaterthan about 99% (w/w), greater than about 99.5% (w/w), or greater thanabout 99.9% (w/w) monoterpene (or sesquiterpene). After storage at 25•Cand 60% relative humidity for at least 3 months, the present compositioncan contain greater than about 98.5% (w/w), greater than about 99%(w/w), greater than about 99.5% (w/w), or greater than about 99.9% (w/w)monoterpene (or sesquiterpene).

The invention also provides for methods of using monoterpenes (orsesquiterpenes) to treat a disease, such as cancer or other nervoussystem disorders. Monoterpenes (or sesquiterpenes) may be administeredalone, or in combination with radiation, surgery or chemotherapeuticagents. The monoterpene or sesquiterpene may also be co-administeredwith antiviral agents, anti-inflammatory agents or antibiotics. Theagents may be administered concurrently or sequentially. Monoterpenes(or sesquiterpenes) can be administered before, during or after theadministration of the other active agent(s).

The monoterpenes (or sesquiterpenes) may also be used as a solvent or apermeation enhancer to deliver a therapeutic agent to the lesion site.For example, monoterpenes (or sesquiterpenes) may be used as a solventor a permeation enhancer to deliver chemotherapeutic agents to tumorcells. The monoterpene or sesquiterpene may also be used as a solventfor vaccines, which may be delivered through any suitable route, such asintranasally.

Monoterpenes (or sesquiterpenes) may be used for the treatment ofnervous system cancers, such as a malignant glioma (e.g., astrocytoma,anaplastic astrocytoma, glioblastoma multiforme), retinoblastoma,pilocytic astrocytomas (grade I), meningiomas, metastatic brain tumors,neuroblastoma, pituitary adenomas, skull base meningiomas, and skullbase cancer. As used herein, the term “nervous system tumors” refers toa condition in which a subject has a malignant proliferation of nervoussystem cells.

Cancers that can be treated by the present monoterpene (orsesquiterpene) compositions include, but are not limited to, lungcancer, ear, nose and throat cancer, leukemia, colon cancer, melanoma,pancreatic cancer, mammary cancer, prostate cancer, breast cancer,hematopoietic cancer, ovarian cancer, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; breast cancer; cervicalcancer; choriocarcinoma; colon and rectum cancer; connective tissuecancer; cancer of the digestive system; endometrial cancer; esophagealcancer; eye cancer; cancer of the head and neck; gastric cancer;intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemiaincluding acute myeloid leukemia, acute lymphoid leukemia, chronicmyeloid leukemia, chronic lymphoid leukemia; liver cancer; lymphomaincluding Hodgkin's and Non-Hodgkin's lymphoma; myeloma; fibroma,neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, andpharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer ofthe respiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; thyroid cancer; uterine cancer; cancer of the urinary system, aswell as other carcinomas and sarcomas. U.S. Pat. No. 7,601,355.

The present invention also provides methods of treating CNS disorders,including, without limitation, primary degenerative neurologicaldisorders such as Alzheimer's, Parkinson's, psychological disorders,psychosis and depression. Treatment may consist of the use of purifiedmonoterpenes or sesquiterpenes alone or in combination with currentmedications used in the treatment of Parkinson's, Alzheimer's, orpsychological disorders. For example, purified monoterpenes orsesquiterpenes may be used as a solvent for the inhalation of currentmedications used in the treatment of Parkinson's, Alzheimer's, orpsychological disorders.

The monoterpene or sesquiterpene may be used in combination withradiation therapy. In one embodiment, the present invention provides fora method of treating tumor cells, such as malignant glioma cells, withradiation, where the cells are treated with an effective amount of amonoterpene, such as perillyl alcohol, and then exposed to radiation.Monoterpene treatment may be before, during and/or after radiation. Forexample, the monoterpene or sesquiterpene may be administeredcontinuously beginning one week prior to the initiation of radiotherapyand continued for two weeks after the completion of radiotherapy. U.S.Pat. Nos. 5,587,402 and 5,602,184.

The present monoterpene or sesquiterpene may be used in combination withat least one therapeutic agents, including, but not limited to,chemotherapeutic agents, immunotherapeutic agents, and antibodies (e.g.,monoclonal antibodies). The anti-cancer agents that may be used incombination with the purified monoterpene or sesquiterpene can have oneor more of the following effects on cancer cells or the subject: celldeath; decreased cell proliferation; decreased numbers of cells;inhibition of cell growth; apoptosis; necrosis; mitotic catastrophe;cell cycle arrest; decreased cell size; decreased cell division;decreased cell survival; decreased cell metabolism; markers of celldamage or cytotoxicity; indirect indicators of cell damage orcytotoxicity such as tumor shrinkage; improved survival of a subject; ordisappearance of markers associated with undesirable, unwanted, oraberrant cell proliferation. U.S. Patent Publication No. 20080275057.

Also encompassed by the present invention are admixtures and/orcoformulations of a monoterpene (or sesquiterpene) and at least onetherapeutic agent, including, but not limited to, a chemotherapeuticagent.

Chemotherapeutic agents include, but are not limited to, DNA alkylatingagents, topoisomerase inhibitors, endoplasmic reticulum stress inducingagents, a platinum compound, an antimetabolite, vincalkaloids, taxanes,epothilones, enzyme inhibitors, receptor antagonists, therapeuticantibodies, tyrosine kinase inhibitors, boron radiosensitizers (i.e.velcade), and chemotherapeutic combination therapies.

In one embodiment, the present invention provides for a method oftreating tumor cells, such as malignant glioma cells, with chemotherapy,where the cells are treated with an effective amount of a monoterpene,such as perillyl alcohol, and then exposed to chemotherapy. Monoterpenetreatment may be before, during and/or after chemotherapy.

DNA alkylating agents are well known in the art and are used to treat avariety of tumors. Non-limiting examples of DNA alkylating agents arenitrogen mustards, such as Mechlorethamine, Cyclophosphamide(Ifosfamide, Trofosfamide), Chlorambucil (Melphalan, Prednimustine),Bendamustine, Uramustine and Estramustine; nitrosoureas, such asCarmustine (BCNU), Lomustine (Semustine), Fotemustine, Nimustine,Ranimustine and Streptozocin; alkyl sulfonates, such as Busulfan(Mannosulfan, Treosulfan); Aziridines, such as Carboquone, ThioTEPA,Triaziquone, Triethylenemelamine; Hydrazines (Procarbazine); Triazenessuch as Dacarbazine and Temozolomide; Altretamine and Mitobronitol.

Non-limiting examples of Topoisomerase I inhibitors include Campothecinderivatives including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin,topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin,lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan,BN-80927, DX-8951f, and MAG-CPT as decribed in Pommier Y. (2006) Nat.Rev. Cancer 6(10):789-802 and U.S. Patent Publication No. 200510250854;Protoberberine alkaloids and derivatives thereof including berberrubineand coralyne as described in Li et al. (2000) Biochemistry39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800;Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine,and fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as describedin Xu (1998) Biochemistry 37(10):3558-3566; and Anthracyclinederivatives including Doxorubicin, Daunorubicin, and Mitoxantrone asdescribed in Foglesong et al. (1992) Cancer Chemother. Pharmacol.30(2):123-125, Crow et al. (1994) J. Med. Chem. 37(19):31913194, andCrespi et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8.Topoisomerase II inhibitors include, but are not limited to Etoposideand Teniposide. Dual topoisomerase I and II inhibitors include, but arenot limited to, Saintopin and other Naphthecenediones, DACA and otherAcridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles,TAS-I03 and other 7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR11576 and other Benzophenazines, XR 5944 and other Dimeric compounds,7-oxo-7H-dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]Perimidines, andAnthracenyl-amino Acid Conjugates as described in Denny and Baguley(2003) Curr. Top. Med. Chem. 3(3):339-353. Some agents inhibitTopoisomerase II and have DNA intercalation activity such as, but notlimited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin,Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin)and Antracenediones (Mitoxantrone and Pixantrone).

Examples of endoplasmic reticulum stress inducing agents include, butare not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, andboron radiosensitizers (i.e. velcade (Bortezomib)).

Platinum based compound which is a subclass of DNA alkylating agents.Non-limiting examples of such agents include Carboplatin, Cisplatin,Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin,Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin. Oncol.201:1232-1237 and in general, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM,CURRENT THERAPY AND NOVEL APPROACHES, in the Series Basic and ClinicalOncology, Angioli et al. Eds., 2004).

“FOLFOX” is an abbreviation for a type of combination therapy that isused to treat colorectal cancer. It includes 5-FU, oxaliplatin andleucovorin. Information regarding this treatment is available on theNational Cancer Institute's web site, cancer.gov, last accessed on Jan.16, 2008.

“FOLFOX/BV” is an abbreviation for a type of combination therapy that isused to treat colorectal cancer. This therapy includes 5-FU,oxaliplatin, leucovorin and Bevacizumab. Furthermore, “XELOX/BV” isanother combination therapy used to treat colorectal cancer, whichincludes the prodrug to 5-FU, known as Capecitabine (Xeloda) incombination with oxaliplatin and bevacizumab. Information regardingthese treatments are available on the National Cancer Institute's website, cancer.gov or from 23 the National Comprehensive Cancer Network'sweb site, nccn.org, last accessed on May 27, 2008.

Non-limiting examples of antimetabolite agents include Folic acid based,i.e. dihydrofolate reductase inhibitors, such as Aminopterin,Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such asRaltitrexed, Pemetrexed; Purine based, i.e. an adenosine deaminaseinhibitor, such as Pentostatin, a thiopurine, such as Thioguanine andMercaptopurine, a halogenated/ribonucleotide reductase inhibitor, suchas Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine:thiopurine, such as Thioguanine; or Pyrimidine based, i.e.cytosine/cytidine: hypomethylating agent, such as Azacitidine andDecitabine, a DNA polymerase inhibitor, such as Cytarabine, aribonucleotide reductase inhibitor, such as Gemcitabine, or athymine/thymidine: thymidylate synthase inhibitor, such as aFluorouracil (5-FU). Equivalents to 5-FU include prodrugs, analogs andderivative thereof such as 5′-deoxy-5-fluorouridine (doxifluroidine),1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda),S-I (MBMS-247616, consisting of tegafur and two modulators, a5-chloro-2,4dihydroxypyridine and potassium oxonate), ralititrexed(tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, asdescribed for example in Papamicheal (1999) The Oncologist 4:478-487.

Examples of vincalkaloids, include, but are not limited to Vinblastine,Vincristine, Vinflunine, Vindesine and Vinorelbine.

Examples of taxanes include, but are not limited to docetaxel,Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An example of anepothilone is iabepilone.

Examples of enzyme inhibitors include, but are not limited tofarnesyltransferase inhibitors (Tipifamib); CDK inhibitor (Alvocidib,Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesteraseinhibitor (Anagrelide; rolipram); IMP dehydrogenase inhibitor(Tiazofurine); and lipoxygenase inhibitor (Masoprocol). Examples ofreceptor antagonists include, but are not limited to ERA (Atrasentan);retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).

Examples of therapeutic antibodies include, but are not limited toanti-HER1/EGFR (Cetuximab, Panitumumab); Anti-HER2/neu (erbB2) receptor(Trastuzumab); Anti-EpCAM (Catumaxomab, Edrecolomab) Anti-VEGF-A(Bevacizumab); Anti-CD20 (Rituximab, Tositumomab, Ibritumomab);Anti-CD52 (Alemtuzumab); and Anti-CD33 (Gemtuzumab). U.S. Pat. Nos.5,776,427 and 7,601,355.

Examples of tyrosine kinase inhibitors include, but are not limited toinhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib,Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTKclass III: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib),PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib,Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib,Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2(Lestaurtinib).

Cetuximab is an example of an anti-EGFR antibody. It is a chimerichuman/mouse monoclonal antibody that targets the epidermal growth factorreceptor (EGFR). Biological equivalent antibodies are identified hereinas modified antibodies and those which bind to the same epitope of theEGFR antigen and produce a substantially equivalent biological responsesuch as, preventing ligand binding of the EGFR, preventing activation ofthe EGFR receptor and the blocking of the downstream signaling of theEGFR pathway resulting in disrupted cell growth.

“Lapatinib” (Tykerb®) is an dual EGFR and erbB-2 inhibitor. Lapatinibhas been investigated as an anticancer monotherapy, as well as incombination with trastuzumab, capecitabine, letrozole, paclitaxel andFOLF1R1 (irinotecan, 5-fluorouracil and leucovorin), in a number ofclinical trials. It is currently in phase III testing for the oraltreatment of metastatic breast, head and neck, lung, gastric, renal andbladder cancer. A chemical equivalent of lapatinib is a small moleculeor compound that is a tyrosine kinase inhibitor (TKI) or alternatively aHER-1 inhibitor or a HER-2 inhibitor. Several TKIs have been found tohave effective antitumor activity and have been approved or are inclinical trials. Examples of such include, but are not limited toZactima (ZD6474), Iressa (gefitinib) and Tarceva (erlotinib), imatinibmesylate (STI571; Gleevec), erlotinib (OSI-1774; Tarceva), canertinib(CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib(BAY 43-9006), sutent (SUI 1248) and lefltmomide (SU101). A biologicalequivalent of lapatinib is a peptide, antibody or antibody derivativethereof that is a HER-1 inhibitor and/or a HER-2 inhibitor. Examples ofsuch include but are not limited to the humanized antibody trastuzumaband Herceptin.

PTK/ZK is a “small” molecule tyrosine kinase inhibitor with broadspecificity that targets all VEGF receptors (VEGFR), theplatelet-derived growth factor (PDGF) receptor, c-KIT and c-Fms. Drevs(2003) Idrugs 6(8):787-794. PTK/ZK is a targeted drug that blocksangiogenesis and lymphangiogenesis by inhibiting the activity of allknown receptors that bind VEGF including VEGFR-I (Flt-1), VEGFR-2(KDR/Flk-1) and VEGFR-3 (Flt-4). The chemical names of PTK/ZK are1-[4-Chloroanilino]-4-[4-pyridylmethyl]phthalazine Succinate or1-Phthalazinamine, N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-butanedioate(1:1). Synonyms and analogs of PTK/TK are known as Vatalanib, CGP79787D,PTK787/ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TKinhibitor, ZK 222584 and ZK.

Chemotherapeutic agents that can be used in combination with thepurified monoterpenes or sesquiterpenes may also include amsacrine,Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide,asparagine depleter Asparaginase/Pegaspargase), Celecoxib, Demecolcine,Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone,Mitoguazone, Mitotane, Oblimersen, Temsirolimus, and Vorinostat.

The compositions and methods of the present invention may be used todecrease the level of the Ras protein. The Ras family is a proteinfamily of small GTPases that are involved in cellular signaltransduction. Activation of Ras signaling causes cell growth,differentiation and survival. Mtations in ras genes can permanentlyactivate it and cause inappropriate transmission inside the cell even inthe absence of extracellular signals. Because these signals result incell growth and division, dysregulated Ras signaling can ultimately leadto oncogenesis and cancer. Activating mutations in Ras are found in20-25% of all human tumors and up to 90% in specific tumor types.Goodsell D S (1999). Downward J., “The molecular perspective: the rasoncogene”. Oncologist 4 (3): 263-4. (January 2003). “Targeting RASsignalling pathways in cancer therapy”. Nat. Rev. Cancer 3 (1): 11-22.Ras family members include, but are not limited to, HRAS; KRAS; NRAS;DIRAS1; DIRAS2; DIRAS3; ERAS; GEM; MRAS; NKIRAS1; NKIRAS2; NRAS; RALA;RALB; RAP1A; RAP1B; RAP2A; RAP2B; RAP2C; RASD1; RASD2; RASL10A; RASL10B;RASL11A; RASL11B; RASL12; REM1; REM2; RERG; RERGL; RRAD; RRAS; and RRAS.Wennerberg K, Rossman K L, Der C J (March 2005). “The Ras superfamily ata glance”. J. Cell. Sci. 118 (Pt 5): 843-6.

The compositions and methods of the present invention may be used toincrease paracellular permeability, for example, paracellularpermeability of endothelial cells or epithelial cells. The presentcompositions and methods may be used to increase blood brain barrierpermeability.

The compositions and methods of the present invention may be used todecrease or inhibit angiogenesis. The present compositions and methodsmay decrease or inhibit production of pro-angiogenic cytokines,including, but not limited to, vascular endothelial growth factor (VEGF)and interleukin 8 (IL8).

The purified monoterpenes or sesquiterpenes may be used in combinationwith angiogenesis inhibitors. Examples of angiogenesis inhibitorsinclude, but are not limited to, angiostatin, angiozyme, antithrombinIII, AG3340, VEGF inhibitors (e.g., anti-VEGF antibody), batimastat,bevacizumab (avastin), BMS-275291, CAI, 2C3, HuMV833 Canstatin,Captopril, carboxyamidotriazole, cartilage derived inhibitor (CDI),CC-5013, 6-O-(chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin,combretastatin A4 Phosphate, Dalteparin, EMD 121974 (Cilengitide),endostatin, erlotinib, gefitinib (Iressa), genistein, halofuginonehydrobromide, Id1, Id3, IM862, imatinib mesylate, IMC-IC11 Inducibleprotein 10, interferon-alpha, interleukin 12, lavendustin A, LY317615 orAE-941, marimastat, mspin, medroxpregesterone acetate, Meth-1, Meth-2,2-methoxyestradiol (2-ME), neovastat, oteopontin cleaved product, PEX,pgment epithelium growth factor (PEGF), platelet factor 4, prolactinfragment, proliferin-related protein (PRP), PTK787/ZK 222584, ZD6474,recombinant human platelet factor 4 (rPF4), restin, squalamine, SU5416,SU6668, SU11248 suramin, Taxol, Tecogalan, thalidomide, thrombospondin,TNP-470, troponin-1, vasostatin, VEG1, VEGF-Trap, and ZD6474.

Non-limiting examples of angiogenesis inhibitors also include, tyrosinekinase inhibitors, such as inhibitors of the tyrosine kinase receptorsFlt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived,fibroblast-derived, or platelet derived growth factors, MMP (matrixmetalloprotease) inhibitors, integrin blockers, pentosan polysulfate,angiotensin II antagonists, cyclooxygenase inhibitors (includingnon-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin andibuprofen, as well as selective cyclooxygenase-2 inhibitors such ascelecoxib and rofecoxib), and steroidal anti-inflammatories (such ascorticosteroids, mineralocorticoids, dexamethasone, prednisone,prednisolone, methylpred, betamethasone).

Other therapeutic agents that modulate or inhibit angiogenesis and mayalso be used in combination with the compounds of the instant inventioninclude agents that modulate or inhibit the coagulation and fibrinolysissystems. Examples of such agents that modulate or inhibit thecoagulation and fibrinolysis pathways include, but are not limited to,heparin, low molecular weight heparins and carboxypeptidase U inhibitors(also known as inhibitors of active thrombin activatable fibrinolysisinhibitor [TAFIa]). U.S. Patent Publication No. 20090328239. U.S. Pat.No. 7,638,549.

The present invention also provides a method of improvingimmunomodulatory therapy responses comprising the steps of exposingcells to an effective amount of a monoterpene or sisquiterpene, such asperillyl alcohol, before or during immunomodulatory treatment. Preferredimmunomodulatory agents are cytokines, such interleukins, lymphokines,monokines, interfereons and chemokines.

This invention further provides for compositions where the purifiedmonoterpene (or sesquiterpene) functions as a solvent or a permeationenhancer. In one aspect, the monoterpene is perillyl alcohol. Examplesof the therapeutic agents are provided infra. The composition mayfurther comprise one or more pharmaceutically acceptable carriers,co-solvents, or other permeation enhancers.

In one embodiment, the composition contains the following components: atherapeutic agent; at least about 0.03% (v/v) of a monoterpene (orsesquiterpene) such as perillyl alcohol; at least about 2.6% (v/v) of aco-solvent which can be 1.3% (v/v) of a polyol such as glycerol or anequivalent thereof; and at least about 1.3% (v/v) of ethanol or anequivalent thereof.

Other permeation enhancers that may be used together with the purifiedmonoterpene (or sesquiterpene) include, but are not limited to, fattyacid esters of glycerin, such as capric, caprylic, dodecyl, oleic acids;fatty acid esters of isosorbide, sucrose, polyethylene glycol;caproyllactylic acid; laureth-2; laureth-2 acetate; laureth-2 benzoate;laureth-3 carboxylic acid; laureth-4; laureth-5 carboxylic acid;oleth-2; glyceryl pyroglutamate oleate; glyceryl oleate; N-lauroylsarcosine; N-myristoyl sarcosine; Noctyl-2-pyrrolidone;lauraminopropionic acid; polypropylene glycol-4-laureth-2; polypropyleneglycol-4-laureth-5dimethyl lauramide; lauramide diethanolamine (DEA),lauryl pyroglutamate (LP), glyceryl monolaurate (GML), glycerylmonocaprylate, glyceryl monocaprate, glyceryl monooleate (GMO) andsorbitan monolaurate. Polyols or ethanol may act as a permeationenhancer or co-solvent. See U.S. Pat. Nos. 5,785,991; 5,843,468;5,882,676; and 6,004,578 for additional permeation enhancers.

Co-solvents are well-known in the art and include, without limitation,glycerol, polyethylene glycol (PEG), glycol, ethanol, methanol,propanol, isopropanol, butanol and the like.

The present composition may be administered by any method known in theart, including, without limitation, intranasal, oral, ocular,intraperitoneal, inhalation, intravenous, ICV, intracisternal injectionor infusion, subcutaneous, implant, vaginal, sublingual, urethral (e.g.,urethral suppository), subcutaneous, intramuscular, intravenous,transdermal, rectal, sub-lingual, mucosal, ophthalmic, spinal,intrathecal, intra-articular, intra-arterial, sub-arachinoid, bronchialand lymphatic administration. Topical formulation may be in the form ofgel, ointment, cream, aerosol, etc; intranasal formulation can bedelivered as a spray or in a drop; transdermal formulation may beadministered via a transdermal patch or iontorphoresis; inhalationformulation can be delivered using a nebulizer or similar device.Compositions can also take the form of tablets, pills, capsules,semisolids, powders, sustained release formulations, solutions,suspensions, elixirs, aerosols, or any other appropriate compositions.

To prepare such pharmaceutical compositions, one or more of the purifiedmonoterpenes (or sesquiterpenes) may be mixed with a pharmaceuticalacceptable carrier, adjuvant and/or excipient, according to conventionalpharmaceutical compounding techniques. Pharmaceutically acceptablecarriers that can be used in the present compositions encompass any ofthe standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, and emulsions, such as an oil/water or water/oilemulsion, and various types of wetting agents. The compositions canadditionally contain solid pharmaceutical excipients such as starch,cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. Liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols. Forexamples of carriers, stabilizers and adjuvants, see Remington'sPharmaceutical Sciences, edited by E. W. Martin (Mack PublishingCompany, 18th ed., 1990). The compositions also can include stabilizersand preservatives.

As used herein, the term “therapeutically effective amount” is an amountsufficient to treat a specified disorder or disease or alternatively toobtain a pharmacological response treating a disorder or disease.Methods of determining the most effective means and dosage ofadministration can vary with the composition used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Treatment dosages generally may be titrated to optimizesafety and efficacy. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician. Suitable dosage formulations and methods of administering theagents can be readily determined by those of skill in the art. Forexample, the composition are administered at about 0.01 mg/kg to about200 mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg toabout 50 mg/kg. When the compounds described herein are co-administeredwith another agent or therapy, the effective amount may be less thanwhen the agent is used alone.

This invention also provides the compositions as described above forintranasal administration. As such, the compositions can furthercomprise a permeation enhancer. Southall et al. Developments in NasalDrug Delivery, 2000. The purified monoterpene (or sesquiterpene) may beadministered intranasally in a liquid form such as a solution, anemulsion, a suspension, drops, or in a solid form such as a powder, gel,or ointment. Devices to deliver intranasal medications are well known inthe art. Nasal drug delivery can be carried out using devices including,but not limited to, intranasal inhalers, intranasal spray devices,atomizers, nasal spray bottles, unit dose containers, pumps, droppers,squeeze bottles, nebulizers, metered dose inhalers (MDI), pressurizeddose inhalers, insufflators, and bi-directional devices. The nasaldelivery device can be metered to administer an accurate effectivedosage amount to the nasal cavity. The nasal delivery device can be forsingle unit delivery or multiple unit delivery. In a specific example,the ViaNase Electronic Atomizer from Kurve Technology (Bethell, Wash.)can be used in this invention (http://www.kurvetech.com). The compoundsof the present invention may also be delivered through a tube, acatheter, a syringe, a packtail, a pledget, a nasal tampon or bysubmucosal infusion. U.S. Patent Publication Nos. 20090326275,20090291894, 20090281522 and 20090317377.

The purified monoterpene (or sesquiterpene) can be formulated asaerosols using standard procedures. The monoterpene (or sesquiterpene)may be formulated with or without solvents, and formulated with orwithout carriers. The formulation may be a solution, or may be anaqueous emulsion with one or more surfactants. For example, an aerosolspray may be generated from pressurized container with a suitablepropellant such as, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen,carbon dioxide, or other suitable gas. The dosage unit can be determinedby providing a valve to deliver a metered amount. Pump spray dispenserscan dispense a metered dose or a dose having a specific particle ordroplet size. As used herein, the term “aerosol” refers to a suspensionof fine solid particles or liquid solution droplets in a gas.Specifically, aerosol includes a gas-borne suspension of droplets of amonoterpene (or sesquiterpene), as may be produced in any suitabledevice, such as an MDI, a nebulizer, or a mist sprayer. Aerosol alsoincludes a dry powder composition of the composition of the instantinvention suspended in air or other carrier gas. Gonda (1990) CriticalReviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburn et al.,(1992) Pharmacol. Toxicol. Methods 27:143-159.

The purified monoterpene (or sesquiterpene) may be delivered to thenasal cavity as a powder in a form such as microspheres delivered by anasal insufflator. The monoterpene (or sesquiterpene) may be absorbed toa solid surface, for example, a carrier. The powder or microspheres maybe administered in a dry, air-dispensable form. The powder ormicrospheres may be stored in a container of the insufflator.Alternatively the powder or microspheres may be filled into a capsule,such as a gelatin capsule, or other single dose unit adapted for nasaladministration.

The pharmaceutical composition can be delivered to the nasal cavity bydirect placement of the composition in the nasal cavity, for example, inthe form of a gel, an ointment, a nasal emulsion, a lotion, a cream, anasal tampon, a dropper, or a bioadhesive strip. In certain embodiments,it can be desirable to prolong the residence time of the pharmaceuticalcomposition in the nasal cavity, for example, to enhance absorption.Thus, the pharmaceutical composition can optionally be formulated with abioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g., highlypurified cationic polysaccharide), pectin (or any carbohydrate thatthickens like a gel or emulsifies when applied to nasal mucosa), amicrosphere (e.g., starch, albumin, dextran, cyclodextrin), gelatin, aliposome, carbamer, polyvinyl alcohol, alginate, acacia, chitosansand/or cellulose (e.g., methyl or propyl; hydroxyl or carboxy;carboxymethyl or hydroxylpropyl).

The composition containing the purified monoterpene (or sesquiterpene)can be administered by oral inhalation into the respiratory tract, i.e.,the lungs.

Typical delivery systems for inhalable agents include nebulizerinhalers, dry powder inhalers (DPI), and metered-dose inhalers (MDI).

Nebulizer devices produce a stream of high velocity air that causes atherapeutic agent in the form of liquid to spray as a mist. Thetherapeutic agent is formulated in a liquid form such as a solution or asuspension of particles of suitable size. In one embodiment, theparticles are micronized. The term “micronized” is defined as havingabout 90% or more of the particles with a diameter of less than about 10μm. Suitable nebulizer devices are provided commercially, for example,by PARI GmbH (Starnberg, Germany). Other nebulizer devices includeRespimat (Boehringer Ingelheim) and those disclosed in, for example,U.S. Pat. Nos. 7,568,480 and 6,123,068, and WO 97/12687. Themonoterpenes (or sesquiterpenes) can be formulated for use in anebulizer device as an aqueous solution or as a liquid suspension.

DPI devices typically administer a therapeutic agent in the form of afree flowing powder that can be dispersed in a patient's air-streamduring inspiration. DPI devices which use an external energy source mayalso be used in the present invention. In order to achieve a freeflowing powder, the therapeutic agent can be formulated with a suitableexcipient (e.g., lactose). A dry powder formulation can be made, forexample, by combining dry lactose having a particle size between about 1μm and 100 μm with micronized particles of the monoterpenes (orsesquiterpenes) and dry blending. Alternatively, the monoterpene can beformulated without excipients. The formulation is loaded into a drypowder dispenser, or into inhalation cartridges or capsules for use witha dry powder delivery device. Examples of DPI devices providedcommercially include Diskhaler (GlaxoSmithKline, Research Triangle Park,N.C.) (see, e.g., U.S. Pat. No. 5,035,237); Diskus (GlaxoSmithKline)(see, e.g., U.S. Pat. No. 6,378,519; Turbuhaler (AstraZeneca,Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769); and Rotahaler(GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365). Further examplesof suitable DPI devices are described in U.S. Pat. Nos. 5,415,162,5,239,993, and 5,715,810 and references therein.

MDI devices typically discharge a measured amount of therapeutic agentusing compressed propellant gas. Formulations for MDI administrationinclude a solution or suspension of active ingredient in a liquefiedpropellant. Examples of propellants include hydrofluoroalklanes (HFA),such as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227), and chlorofluorocarbons,such as CCl₃F. Additional components of HFA formulations for MDIadministration include co-solvents, such as ethanol, pentane, water; andsurfactants, such as sorbitan trioleate, oleic acid, lecithin, andglycerin. (See, for example, U.S. Pat. No. 5,225,183, EP 0717987, and WO92/22286). The formulation is loaded into an aerosol canister, whichforms a portion of an MDI device. Examples of MDI devices developedspecifically for use with HFA propellants are provided in U.S. Pat. Nos.6,006,745 and 6,143,227. For examples of processes of preparing suitableformulations and devices suitable for inhalation dosing see U.S. Pat.Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO 99/53901, WO00/61108, WO 99/55319 and WO 00/30614.

The monoterpenes (or sesquiterpenes) may be encapsulated in liposomes ormicrocapsules for delivery via inhalation. A liposome is a vesiclecomposed of a lipid bilayer membrane and an aqueous interior. The lipidmembrane may be made of phospholipids, examples of which includephosphatidylcholine such as lecithin and lysolecithin; acidicphospholipids such as phosphatidylserine and phosphatidylglycerol; andsphingophospholipids such as phosphatidylethanolamine and sphingomyelin.Alternatively, cholesterol may be added. A microcapsule is a particlecoated with a coating material. For example, the coating material mayconsist of a mixture of a film-forming polymer, a hydrophobicplasticizer, a surface activating agent or/and a lubricantnitrogen-containing polymer. U.S. Pat. Nos. 6,313,176 and 7,563,768.

Because of their ability to easily penetrate the dermis, monoterpenesmay also be used alone or in combination with other chemotherapeuticagents via topical application for the treatment of localized cancerssuch as breast cancer or melanomas. As a transdermal delivery agent,monoterpenes may also be used in combination with narcotics oranalgesics for transdermal delivery of pain medication.

This invention also provides the compositions as described above forocular administration. As such, the compositions can further comprise apermeation enhancer. For ocular administration, the compositionsdescribed herein can be formulated as a solution, emulsion, suspension,etc. A variety of vehicles suitable for administering compounds to theeye are known in the art. Specific non-limiting examples are describedin U.S. Pat. Nos. 6,261,547; 6,197,934; 6,056,950; 5,800,807; 5,776,445;5,698,219; 5,521,222; 5,403,841; 5,077,033; 4,882,150; and 4,738,851.

The monoterpenes (or sesquiterpenes) can be given alone or incombination with other drugs for the treatment of the above diseases fora short or prolonged period of time. The present compositions can beadministered to a mammal, preferably a human. Mammals include, but arenot limited to, murines, rats, rabbit, simians, bovines, ovine, porcine,canines, feline, farm animals, sport animals, pets, equine, andprimates.

The present invention further provides an article of manufacture (suchas a kit) comprising the purified monoterpene (or sesquiterpene)formulated for intranasal administration, and a device for intranasaladministration of the purified monoterpene (or sesquiterpene). Thedevice for intranasal administration may be an intranasal spray device,an atomizer, a nebulizer, a metered dose inhaler (MDI), a pressurizeddose inhaler, an insufflator, an intranasal inhaler, a nasal spraybottle, a unit dose container, a pump, a dropper, a squeeze bottle, or abi-directional device. The article of manufacture can contain printedmatter indicating purified monoterpene (or sesquiterpene) is to be usedto treat a disease, such as cancer or other nervous system disorders.The printed matter may state that the monoterpenes (or sesquiterpenes)may be administered alone, or in combination with radiation, surgery orchemotherapeutic agents. The monoterpene or sesquiterpene may also beco-administered with antiviral agents, anti-inflammatory agents orantibiotics. The agents may be administered concurrently orsequentially.

The invention also provides a method for inhibiting the growth of a cellin vitro, ex vivo or in vivo, where a cell, such as a cancer cell, iscontacted with an effective amount of the purified monoterpene (orsesquiterpene) as described herein. The present compositions and methodsmay be used to inhibit the growth of a cell that is resistant to achemotherapeutic agent. For example, the present compositions andmethods may be used to inhibit the growth of a temozolomide-resistantcell.

Pathological cells or tissue such as hyperproliferative cells or tissuemay be treated by contacting the cells or tissue with an effectiveamount of a composition of this invention. The cells, such as cancercells, can be primary cancer cells or can be cultured cells availablefrom tissue banks such as the American Type Culture Collection (ATCC).The pathological cells can be cells of a systemic cancer, gliomas,meningiomas, pituitary adenomas, or a CNS metastasis from a systemiccancer, lung cancer, prostate cancer, breast cancer, hematopoieticcancer or ovarian cancer. The cells can be from a vertebrate, preferablya mammal, more preferably a human. U.S. Patent Publication No.2004/0087651. Balassiano et al. (2002) Intern. J. Mol. Med. 10:785-788.Thorne, et al. (2004) Neuroscience 127:481-496. Fernandes, et al. (2005)Oncology Reports 13:943-947. Da Fonseca, et al. (2008) SurgicalNeurology 70:259267. Da Fonseca, et al. (2008) Arch. Immunol. Ther. Exp.56:267-276. Hashizume, et al. (2008) Neuroncology 10:112-120.

Cancer stem cells (CSCs) or tumour initiating cells are immature cellswith stem cell features such as self-renewal. However, self-renewal isexacerbated in CSCs. Reya et al., Stem cells, cancer, and cancer stemcells. Nature. 2001, 414(6859):105-11. Additionally, glioma CSCs areresistant to chemo- and radio-therapy. Bao et al., Glioma stem cellspromote radioresistance by preferential activation of the DNA damageresponse. Nature. 2006, 444(7120):756-60. Rich et al., Chemotherapy andcancer stem cells. Cell Stem Cell. 2007; 1(4):353-5. The presentcompositions and methods may be used to inhibit the growth of a cancerstem cell, including, but not limited to, a glioblastoma cancer stemcell.

In vitro efficacy of the present composition can be determined usingmethods well known in the art. For example, the cytoxicity of thepresent monoterpene (or sesquiterpene) and/or the therapeutic agents maybe studied by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide] cytotoxicity assay. MTT assay is based on the principle ofuptake of MTT, a tetrazolium salt, by metabolically active cells whereit is metabolized into a blue colored formazon product, which can beread spectrometrically. J. of Immunological Methods 65: 55 63, 1983. Thecytoxicity of the present monoterpene (or sesquiterpene) and/or thetherapeutic agents may be studied by colony formation assay. Functionalassays for inhibition of VEGF secretion and IL-8 secretion may beperformed via ELISA. Cell cycle block by the present monoterpene (orsesquiterpene) and/or the therapeutic agents may be studied by standardpropidium iodide (PI) staining and flow cytometry. Invasion inhibitionmay be studied by Boyden chambers. In this assay a layer ofreconstituted basement membrane, Matrigel, is coated onto chemotaxisfilters and acts as a barrier to the migration of cells in the Boydenchambers. Only cells with invasive capacity can cross the Matrigelbarrier. Other assays include, but are not limited to cell viabilityassays, apoptosis assays, and morphological assays.

The following examples are presented for the purposes of illustrationonly and are not limiting the invention.

Example 1 (S)-Perillyl Alcohol Purification Via 3,5-DinitrobenzoateEster

(S)-Perillyl alcohol can be purified directly from natural products, orbe obtained by synthetic modification of natural products such asbeta-pinene (extracted from pine trees) by oxidation and rearrangement(Scheme 1).

Such sources of (S)-perillyl alcohol are inevitably contaminated byisomers of the target compound which are very similar in physicochemicalproperties, and therefore, are difficult to remove by conventionalmethods of purification such as fractional distillation orchromatography.

In this Example, in order to purify (S)-perillyl alcohol from thecontaminants that typically accompany it from natural product and/orsynthetic sources, perillyl alcohol was first derivatized as its3,5-dinitrobenzoate ester, which was separated from contaminants byconventional crystallization. Once the derivatized (S)-perillyl alcoholhas been purified by crystallization it can then be hydrolyzed torecover the purified (S)-perillyl alcohol (Scheme 2). The purified(S)-perillyl alcohol prepared in this way has a purity greater thanabout 99%.

Synthesis of 3,5-Dinitrobenzoic acid 4-(S)-isopropenylcyclohex-1-enylmethyl ester (Compound 3)

Triethyl amine (12.2 mL, 87.5 mmol) was added to a mixture of(S)-perillyl alcohol (1, 89.5% 10.0 g, 58.7 mmol) in dichloromethane (70ml) over a period of 0.25 h while maintaining the temperature below 15°C. The reaction mixture was stirred for 30 min at room temperature. Asolution of 3,5-dinitro benzoyl chloride (Compound 2, 14.23 g, 61.7mmol) dissolved in dichloromethane (30 mL) was added over a period of0.5 h while keeping the temperature below 15° C. The reaction mixturewas allowed to warm to room temperature and then stirred for 3.0 h. Thereaction mixture was quenched with water (75 mL) and the organic layerwas separated. The aqueous layer was extracted with dichloromethane (50mL). The combined organic layer was washed with water (2×100 mL) anddried over sodium sulphate (25 g). The filtered organic layer wasconcentrated and the resulting residue was crystallized from diisopropylether (200 mL) to get pure compound 3 (Weight: 14.45 g). The motherliquor was concentrated to half of its volume and 2.1 g was obtained asa second crop. (Total yield: 81.2%, Purity: 99.4% by HPLC.)

Hydrolysis of 3,5-Dinitrobenzoic acid 4-(S)-isopropenylcyclohex-1-enylmethyl ester (Compound 3)

Aqueous sodium hydroxide (3.23 g, 80.0 mmol, dissolved in 28 mL ofwater) was added to an ice cold solution of 3,5-Dinitro-benzoic acid4-isopropenyl-cyclohex-1-enylmethyl ester (14.0 g, 40.4 mmol) inmethanol (140 mL) over a period of 0.25 h. The reaction mixture wasallowed to warm to room temperature and then stirred for 3.0 h. Themethanol was concentrated under vacuum and the resulting residue wassuspended in water (60 mL) and extracted with ethyl acetate (2×100 mL).The organic layer was washed with water (2×100 mL) followed by brine(15%, 100 mL) and dried over sodium sulphate (30 g). The filteredorganic layer was concentrated under vacuum to get pure (S)-perillylalcohol (Weight: 5.84 g, Yield: 95% Purity: 99.4% by GC).

Example 2 Synthesis of 3,5-Dinitrobenzoic acid 4(S)-isopropenylcyclohex-1-enylmethyl ester (Compound 3) and Purification by PreparativeChromatography

Triethyl amine (5.3 mL, 38.0 mmol) was added to a mixture of(S)-perillyl alcohol (89.5% 5.0 g, 29.3 mmol) in dichloromethane (40 ml)over a period of 0.25 h while maintaining the temperature below 15° C.The reaction mixture was stirred for 30 min at room temperature. Asolution of 3,5-dinitro benzoyl chloride (7.43 g, 32.2 mmol) dissolvedin dichloromethane (15 mL) was added over a period of 0.5 h whilemaintain the temperature between 15-20° C. The reaction mixture wasallowed to warm to room temperature and then stirred for 3.0 h. Thereaction mixture was quenched with water (40 mL) and the organic layerwas separated. The aqueous layer was extracted with dichloromethane (25mL). The combined organic layer was washed with water (2×50 mL) anddried over sodium sulphate (20 g). The filtered organic layer wasconcentrated under vacuum to give a residue which was purified by columnchromatography. Column dimensions were as follows: diameter: 2.5 cm,height: 30 cm, silica: 200 mesh. The column was eluted withhexanes:ethyl acetate (98:2, 200 mL) followed by hexanes: ethyl acetate(95:5). Based on TLC analysis of the fractions (solvent system; hexanes:ethyl acetate (90:10)), the hexanes: ethyl acetate (95:5) fractions werecombined and concentrated under vacuum to give a solid. (Weight: 7.9 gYield: 78%.)

Example 2 Synthesis of 4-Nitrobenzoic acid 4(S)-isopropenylcyclohex-1-enylmethyl Ester (Scheme 3)

Triethyl amine (5.92 mL, 42.4 mmol) was added to a mixture of(S)-perillyl alcohol (5.0 g, 32.8 mmol) in dichloromethane (30 ml) overa period of 0.25 h while maintaining the temperature below 15° C. Thereaction mixture was stirred for 30 min at room temperature. A solutionof 4-nitrobenzoyl chloride (6.39 g, 34.4 mmol) dissolved indichloromethane (30 mL) was added over a period of 0.5 h while keepingthe temperature below 15° C. The reaction mixture was allowed to warm toroom temperature and then stirred for 3.0 h. The reaction mixture wasquenched with water (50 mL) and the organic layer was separated. Theaqueous layer was extracted with dichloromethane (25 mL). The combinedorganic layer was washed with water (2×50 mL) and dried over sodiumsulphate (20 g). The filtered organic layer was concentrated to give anoil (Weight: 8.9 g, yield: 90%).

Example 3 Synthesis of 4-chlorobenzoic acid 4(S)-isopropenylcyclohex-1-enylmethyl Ester (Scheme 4)

Triethyl amine (2.85 mL, 20.5 mmol) was added to a mixture of(S)-perillyl alcohol (2.5 g, 16.4 mmol) in dichloromethane (25 ml) overa period of 0.25 h while maintaining the temperature below 15° C. Thereaction mixture was stirred for 30 min at room temperature. A solutionof 4-chlorobenzoyl chloride (3.01 g, 17.2 mmol) dissolved indichloromethane (10 mL) was added over a period of 0.5 h while keepingthe temperature below 15° C. The reaction mixture was allowed to warm toroom temperature and then stirred for 3.0 h. The reaction mixture wasquenched with water (30 mL) and the organic layer was separated. Theaqueous layer was extracted with dichloromethane (25 mL). The combinedorganic layer was washed with water (2×30 mL) and dried over sodiumsulphate (15 g). The filtered organic layer was concentrated to give anoil (Weight: 3.8 g, yield: 81.7%).

Example 4 Synthesis of 3,4,5-trimethoxybenzoic acid 4(S)-isopropenylcyclohex-1-enylmethyl Ester (Scheme 5)

Triethyl amine (2.85 mL, 20.5 mmol) was added to a mixture of(S)-perillyl alcohol (2.5 g, 16.4 mmol) in dichloromethane (25 ml) overa period of 0.25 h while maintaining the temperature below 15° C. Thereaction mixture was stirred for 30 min at room temperature. A solutionof 3,4,5-trimethoxybenzoyl chloride (3.97 g, 17.2 mmol) dissolved indichloromethane (10 mL) was added over a period of 0.5 h while keepingthe temperature below 15° C. The reaction mixture was allowed to warm toroom temperature and then stirred for 3.0 h. The reaction mixture wasquenched with water (30 mL) and the organic layer was separated. Theaqueous layer was extracted with dichloromethane (25 mL). The combinedorganic layer was washed with water (2×30 mL) and dried over sodiumsulphate (15 g). The filtered organic layer was concentrated to give anoil (Weight: 4.8 g, yield: 84.6%).

Example 5 Synthesis of 4-trimethoxybenzoic acid 4(S)-isopropenylcyclohex-1-enylmethyl Ester (Scheme 6)

Triethyl amine (2.97 mL, 21.3 mmol) was added to a mixture of(S)-perillyl alcohol (2.5 g, 16.4 mmol) in dichloromethane (25 ml) overa period of 0.25 h while maintaining the temperature below 15° C. Thereaction mixture was stirred for 30 min at room temperature. A solutionof 4-methoxybenzoyl chloride (2.94 g, 17.2 mmol) dissolved indichloromethane (10 mL) was added over a period of 0.5 h while keepingthe temperature below 15° C. The reaction mixture was allowed to warm toroom temperature and then stirred for 3.0 h. The reaction mixture wasquenched with water (30 mL) and the organic layer was separated. Theaqueous layer was extracted with dichloromethane (25 mL). The combinedorganic layer was washed with water (2×30 mL) and dried over sodiumsulphate (15 g). The filtered organic layer was concentrated to give anoil (Weight: 4.1 g, yield: 87%).

Example 6 Functional Assays of Perillyl Alcohol In Vitro

(S)-perillyl alcohol with greater than 98.5% purity was compared tocommercial (S)-perillyl alcohol purchased from Sigma Chemicals (96%purity) at concentrations 650 μM or 1.3 mM using morphological assays,cytotoxicity assays with MTT to quantify cytotoxicity for dispersedcells, and colony formation assays (CFA). Morphological assaysdemonstrated that both the purified (S)-perillyl alcohol (greater than98.5% purity) and the (S)-perillyl alcohol from Sigma (96% purity) hadcytotoxicity activity on A172 malignant glioma cells within 12 hours.Both drugs induced cell rounding and detachment from the plate (FIG. 1).MTT assays showed that purified (S)-perillyl alcohol exhibited bettercytotoxicity on U87 human malignant glioma cells than the less pure(S)-perillyl alcohol from Sigma (FIG. 2). The numeric difference incytotoxic effect between the purified (S)-perillyl alcohol and Sigma(S)-perillyl alcohol is much greater than the numeric difference in(S)-perillyl alcohol purity. Compared to Sigma (S)-perillyl alcohol, thepurity increase for the purified (S)-perillyl alcohol is about 2.6%; thecytotoxic property of the purified (S)-perillyl alcohol increased about125%.

FIG. 3A shows the results of the MTT cytotoxicity assays demonstratingthe efficacy of purified POH in killing various kinds of human gliomacells, including U251, U87, LN299 and A172 cells. FIG. 3B shows theresults of the MTT assays performed using temozolomide (TMZ) sensitiveU251 glioma cells and temozolomide resistant U251 cell line (U251/TR1and U251/TR2) over 24 hours using purified POH having a purity of about98.7%. Purified POH is effective in killing both U251 human glioma cells(temozolomide-sensitive) and U251 temozolomide-resistant cells. Theefficacy of purified POH on U251 temozolomide-resistant cells is alsodemonstrated by the colony formation assays shown in FIG. 3C.

POH has also been demonstrated to inhibit glioma invasion through aBoyden matrigel chamber, suggesting that it also has anti-invasionproperties.

POH was found to be anti-angiogenic, as POH inhibited production of thepro-angiogenic cytokines vascular endothelial growth factor (VEGF) andinterleukin 8 (IL8) by glioma cells. Functional assays for inhibition ofVEGF secretion and IL-8 secretion by POH have been performed via ELISAassays (FIG. 9). POH has been shown to inhibit G1 cell cycle arrest.Pyrko et al. The unfolded protein response regulator GRP78/BiP as anovel target for increasing chemosensitivity in malignant gliomas.Cancer Res 67(20):9809-16, 2007. Cell cycle analysis after POH treatmentwill be performed using standard propidium iodide (PI) staining and flowcytometry. In addition, it may overcome the immunosuppressive functionsof transforming growth factor beta-2 (TGFβ-2) secreted by glioma cells.

U251 glioma cells were treated with Sigma POH or purified POH for 24hours, then Western blot was performed. The results (FIG. 12)demonstrate that POH treatment decreased K-Ras and H-Ras expression.

Example 7 Combination of POH and Radiation In Vitro

Purified (S)-POH having greater than 98.5% purity was assessed as aradiation sensitizer in human A172 glioma cells using the colonyformation assays. Cells were treated with 325 μM or 650 μM POH prior toradiation at the doses of 2, 5 or 10 Grays (Gy). POH was shown to actsynergistically with radiation in cell killing (FIG. 4A). This wasconfirmed for another human glioma cell line (U251) and also for amelanoma cell line (B16-F1) (FIG. 4B).

Example 8 Combination of Perillyl Alcohol with Chemotherapeutic AgentsIn Vitro

Cytotoxicity assays were carried out after cells were treated with POHin combination with temozolomide (TMZ), the standard alkylating agentused in the treatment of malignant gliomas. POH and TMZ was at leastadditive in cytotoxicity (or synergistic) (FIG. 5C). This effect wasalso seen in cell lines that were TMZ resistant.

Similarly, POH was at least additive (or synergistic) withdimethyl-celecoxib (DMC), determined by MTT cytotoxicity assays onvarious human glioma cell lines (FIG. 5A) and colony formation assays.POH was at least additive (or synergistic) with Nelfinavir (NFV),determined by MTT cytotoxicity assays (FIG. 5B). Pyrko et al. HIV-1protease inhibitors nelfinavir and atazanavir induce malignant gliomadeath by triggering endoplasmic reticulum stress. Cancer Res 67(22):10920-10928, 2007.

POH was also tested in combination with rolipram, a type IVphosphodiesterase which was demonstrated to cause differentiation andapoptosis in glioma cells. When cells were treated with rolipram alone,apoptosis occurred only after 48 hours post the addition of highconcentrations of rolipram (e.g., 1 mM). Synergistic cell killing byrolipram and POH occurred within a much shorter time period, i.e., 6hours after the addition of the drugs. Chen et al. The type IVphosphodiesterase inhibitor rolipram induces expression of the cellcycle inhibitors p21 Cip 1 and p27Kip1, resulting in growth inhibition,increased differentiation, and subsequent apoptosis of malignant A-12glioma cells. Cancer Biology & Therapy, Vol. 1:3, 268-276, 2003.

Example 9 POH Increases Paracellular Permeability and ER Stress

FIG. 6 shows the results of propidium iodide (PI) staining of A172,A375, U251 human malignant glioma cells, and B16-F1 melanoma cells. 650μM POH having a purity greater than 98.5% increased paracellularpermeability to a greater extent than 0.04% Triton X, the positivecontrol.

Transepithelial electrical resistance (TEER) assays were performedassessing the ability of POH to increase paracellular permeability (FIG.7A). In these experiments, brain endothelial cells (BECs) were used asan in vitro blood brain barrier model. FIG. 7B shows that POH decreasedTEER, suggesting POH treatment increased paracellular permeability. TEERdecreased when cells were treated by 0.03% POH for 20 hours compared tomedium only, 0.01% POH treatment, and 0.02% POH treatment. The increasein TEER in each group over time corresponds to increased confluency ofthe cells in culture.

FIG. 8 shows that purified (S)-perillyl alcohol increases epithelialparacellular permeability. This result suggests that purified(S)-perillyl alcohol may increase BBB permeability. 2.5×10⁵ Madin-DarbyCanine Kidney (MDCK) cells were seeded in each well of the cell cultureplate and cultured for 5 days. The cell culture media were changed 3days after seeding and 24 hours before being treated with POH. MDCKcells in a monolayer were treated overnight with varying concentrationsof purified (S)-perillyl alcohol having a purity greater than 98.5%.Fluorescein labeled antibodies were then added to the cells andincubated for 2 hours. The amount of labeled antibodies that crossed thecell monolayer was then quantitated by fluorescence.

POH also induced endoplasmic reticulum (ER) stress via interaction withcytosolic proteins. POH up-regulated the levels of ER stress markerglucose-regulated protein 78 (GRP78) and apoptosis marker CCAAT/enhancerbinding protein (CHOP) in various human glioma cell lines, includingU251, U87 and A172 cells (FIG. 10). GRP78 is an anti-apoptotic proteinthe level of which is increased in response to stress. CHOP is aproapoptotic protein signaling for apoptosis. Pyrko et al. The unfoldedprotein response regulator GRP78/BiP as a novel target for increasingchemosensitivity in malignant gliomas. Cancer Research 67(20):9809-16,2007.

U251 TMZ-sensitive and TMZ-resistant (U251/TR1, U251/TR2) cells weretreated with Sigma POH (1.5 mM) or purified POH (1.5 mM) for 20 hours,then Western blot was performed. The results show that Sigma POH andpurified POH increased expression of glucose-regulatory protein 78(GRP-78) and the apoptosis marker CHOP, suggesting increased endoplasmicreticulum (ER) stress after treatment (FIG. 11).

Example 10 POH Effectively Kills Cancer Stem Cells

FIG. 13 shows the results of the MTT assay performed using glioblastomacancer stem cell line USC-02, glioma tumour cells (U251 cells) or normalstem cells (mSVZ: mouse SVZ stem/progenitor cells) treated with POH for48 hrs. The cancer stem cells were more sensitive to POH than gliomatumour cells (U251 cells) or normal stem cells (mSVZ: mouse SVZstem/progenitor cells). FIG. 14 shows that the glioma tumor cells (U251cells) were more sensitive to POH than normal stem cells (brainendothelial cells (BECs) and astrocytes). FIG. 15A shows the results ofthe MTT cytotoxicity assays demonstrating that the cancer stem cellsUSC-04 were more sensitive to POH (EC₅₀ on USC-04=0.3 mM) than gliomatumor cells (U251 cells). FIG. 15B shows the results of the MTTcytotoxicity assays demonstrating that the cancer stem cells USC-04 weremore resistant to DMC (EC₅₀ on USC-04=78 μM) than glioma tumor cells(U251 cells). Verapamil is used as an inhibitor of drug efflux pumpproteins such as P-glycoprotein. The results suggest that DMC resistanceof the cancer stem cells was not due to drug efflux via P-glycoprotein.

FIG. 16 shows the results of sphere formation assay (SFA) demonstratingthat POH decreased the number of spheres formed in glioblastoma cancerstem cells (USC04). USC04 cells were incubated with varyingconcentrations of POH (0-1.5 mM) for 6 days.

FIG. 17 shows that POH decreased H-ras production in glioblastoma cancerstem cells. Two glioblastoma cancer stem cell lines (USC-02, USC-04)were treated by POH for 24 hours. USC-02 and USC-04 are two independentprimary cancer stem cell lines isolated from glioblastoma tissue fromtwo different patients.

Example 11 In Vivo Animal Studies

The purified POH prepared by the process in Example 1 will be placedinto an intranasal inhaler (e.g., the ViaNase Electronic Atomizer fromKurve Technology (Bethell, Wash.)). The intranasal delivery system fromKurve Technology is capable of accurately delivering a pre-determineddrug volume (e.g., from 0.2-6 mL). The device is loaded and cleaned inthe same manner as a pulmonary nebulizer. The device can deliver thedrug to the olfactory region in bench testing, in animals and humans.

Male athymic nu/nu mice (6-8 weeks old) will be employed for thisresearch. Rodent subcutaneous/intracranial glioma model can beestablished as follows. Six to eight week old athymic nu/nu mice will beanesthesized with intraperitoneal injections of ketamine (80 mg/kg) andxylazine (10 mg/kg). For the intracranial glioma model, the mice areplaced into a stereotactic head frame (Harvard Apparatus), and localanesthetic (0.2 cc of 0.25% xylocaine) is injected into the rightfrontal scalp. A knife blade is used to make a small incision, and adrill bit is used to make a small opening in the right frontal skull atthe level of the coronal suture. Glioma cells (1×10⁵ cells/10 μl), forexample, U-87 human glioma cells, will be loaded into a calibratedHamilton syringe. The needle tip will be placed precisely into the rightfrontal lobe of the rat, and cells will be slowly injected using acontrol push from the Hamilton syringe. After the injection is finished,the syringe and needle will be removed, and the wound closed.

Two weeks after surgical implantation, the mice will be divided into 4groups (6 mice/group) and will be treated, respectively, with: salinedrops alone (control), crude POH from Sigma (0.03%, 50 ul/drop, one dropper nostril), POH (purified to greater than 98.5% purity; 0.03%, 50ul/drop, one drop per nostril), and TMZ (5 mg/kg, oral gavage). TMZserves as the positive control.

Brains will be harvested, and tumor size determined. Survival curveswill be constructed by following the mice until they developneurological deficits. Our experience has been that survival is aboutfour weeks after implantation for untreated mice, and up to 8 weeks formice treated with TMZ.

We will also use an immune-competent syngeneic rat model where RG2 ratglioma cells (1×10⁵ cells/10 ul) will be implanted into the rightfrontal lobe of Fisher 344 rats. Rats will be divided into the same 4groups as above. We will also examine the anti-invasion properties ofPOH using the rat RG2 model, because the RG2 cells can freely migrate,and thus, invade in the rat parenchyma.

Example 12 Patient Studies

In a recent clinical research in Brazil, intranasal delivery of perillylalcohol in patients with recurrent malignant gliomas resulted inregression or stabilization of the disease, with 50% of the 140 treatedpatients achieving 6 month progression-free period and several patientsenjoying as many as 3 years of disease remission. Furthermore, sideeffects from the treatment were almost non-existent. Da Fonseca et al.Correlation of tumor topography and peritumoral edema of recurrentmalignant gliomas with therapeutic response to intranasal administrationof perillyl alcohol. Invest New Drugs 2009, Jan. 13.

We will deliver the purified POH (having greater than 98.5% purity)intranasally to patients suffering from malignant gliomas. Toinvestigate whether POH can be delivered directly to the brain tumorcells, the distribution of the purified POH will be studied bydelivering ¹¹C labeled-POH to the patients, followed by positronemission tomography (PET) imaging. The patients will then undergo alimited therapeutic trial using escalating doses of inhalational POH.The patients will be dose escalated using groups of three, with eachgroup receiving intranasal purified POH (with purity greater than 98.5%)at 0.05% (w/v), 1% (w/v), 1.5% (w/v), 2% (w/v), 2.5% (w/v). The 2% (w/v)is what is currently used in Brazil. Delivery will be via the ViaNasenasal inhaler and will be given three times per day. PET ImagingStudies. Ten patients with pathologically confirmed malignant gliomawill be scanned following intranasal inhalation of 5-10 mCi of the¹¹C-POH formulation using a Siemens Biograph TruePoint HD PET/CTscanner. Static imaging will begin at 30 minutes following inhalationusing 10-minute acquisition in a single bed position overlying thecranium. Subsequent serial acquisitions will occur at 30-minuteintervals for 2 hours to assess progressive accumulation in brain andtumor tissue. Depending on patient compliance and levels of remainingand accumulated activity, we will attempt to image beyond 2 hours.Co-registered PET/CT images will be compared with contrast enhanced MRIstudies on all patients to assess correlation of activity accumulationwith enhancement patterns.

The scope of the present invention is not limited by what has beenspecifically shown and described hereinabove. Those skilled in the artwill recognize that there are suitable alternatives to the depictedexamples of materials, configurations, constructions and dimensions.Numerous references, including patents and various publications, arecited and discussed in the description of this invention. The citationand discussion of such references is provided merely to clarify thedescription of the present invention and is not an admission that anyreference is prior art to the invention described herein. All referencescited and discussed in this specification are incorporated herein byreference in their entirety. Variations, modifications and otherimplementations of what is described herein will occur to those ofordinary skill in the art without departing from the spirit and scope ofthe invention. While certain embodiments of the present invention havebeen shown and described, it will be obvious to those skilled in the artthat changes and modifications may be made without departing from thespirit and scope of the invention. The matter set forth in the foregoingdescription and accompanying drawings is offered by way of illustrationonly and not as a limitation.

What is claimed is:
 1. A process for purifying perillyl alcoholcomprising the steps of: (a) derivatizing a mixture comprising perillylalcohol to form a perillyl alcohol derivative, wherein the perillylalcohol derivative is a benzoate ester derivative of perillyl alcohol;(h) crystallizing the perillyl alcohol derivative; (c) separating theperillyl alcohol derivative crystals from the mixture; (d) releasing theperillyl alcohol from the separated perillyl alcohol derivative fromstep (c); and, (e) isolating the perillyl alcohol from step (d).
 2. Theprocess of claim 1, wherein the benzoate ester is 3,5-dinitrobenzoateester.
 3. The process of claim 1, wherein the isolated perillyl alcoholhas a purity of greater than about 98.5% (w/w).
 4. The process of claim3, wherein the isolated perillyl alcohol has a purity of greater thanabout 99.0% (w/w).
 5. The process of claim 1, wherein the mixturefurther comprises natural-product-derived or other impurities.
 6. Theprocess of claim 4, wherein the isolated perillyl alcohol has a purityof greater than about 99.5% (w/w).
 7. The process of claim 1, whereinthe perillyl alcohol is (S)-perillyl alcohol.